Abstract: The fungi Cryptococcus neoformans and C. gattii cause cryptococcal meningitis (CM) and pulmonary cryptococcosis, which mainly occurs in immunocompromised people, such as those with HIV, severe combined immunodeficiency, or post organ-transplant status. Recent outbreaks of infections of C. gattii in immune competent people raise significant concerns about the overall threat of cryptococcal species to public health. There are over 1 million annual cases of CM worldwide, with estimated deaths of 700,000 per year. Treatment of CM has become increasingly difficult with a mortality rate over 50%, due to the emergence of drug-resistant strains, making the development of new treatments for cryptococcal infections imperative. Cryptococcal fungi contain protein self-splicing elements, called inteins, which are internal protein elements that self-excise from their host proteins and catalyze ligation of the flanking sequences (exteins) with a natural peptide bond. Because inteins do not exist in multi-cellular organisms, such as humans, and often disrupt the functions of critical microbial genes, they are attractive drug targets. Cryptococcal species have inteins in the essential gene, Prp8, a component of the spliceosome. Intein inhibitors have many advantages over traditional anti-fungal drugs. Because the Prp8 inteins of cryptococcal species share high sequence similarity and have similar splicing mechanisms, it is highly possible that a single intein splicing inhibitor can inhibit the splicing activities of all Prp8 inteins. Thus, Prp8 intein splicing inhibitors will be ?broad-spectrum? against all Prp8 intein-containing fungal pathogens. On the other hand, because neither human nor microbes normally associated with humans have inteins, intein inhibitors would be also function as ?narrow-spectrum? antifungals specific only for intein-containing pathogens such as cryptococcal species. In addition, inteins as novel drug targets have an inhibition mechanism different from those of all known drugs. Thus, intein inhibitors will provide a potentially fresh approach and may synergize with existing drugs in treating the infected. In our preliminary results, cisplatin was found not only to inhibit the intein splicing in vitro, but also to reduce the fungal burden in vivo. The major goal of this proposal is to develop and perform high throughput screening assays to identify and characterize compounds that inhibit the Prp8 intein splicing by a combination of biophysical, biochemical, computational, pharmacological, medicinal, cellular and in vivo approaches. Hit compounds with activity better than the existing drugs will be tested for reduction of growth of cryptococcal species using various assays and resistant variants. This study will generate information on inhibitor-intein interaction at structurally significant sites and potentially lead to novel therapies for cryptococcal infections.